Apolipoprotein E: Remarkable Protein Sheds Light on Cardiovascular and Neurological Diseases

Abstract

Apolipoprotein E (apoE)6 is a relatively small protein (34 kDa, 299 amino acids) that has been slowly revealing its biologic secrets in cardiovascular and neurological diseases over the past 45 years since it was first observed as a protein constituent of plasma lipoproteins. ApoE is encoded by a single gene on chromosome 19 [apolipoprotein E (APOE)]. Its seemingly complex isoform polymorphism involves 3 isoforms that differ by single amino acid interchanges at residues 112 and 158, resulting from single base changes in the APOE gene. ApoE3, the most common isoform (ε3 prevalence 65%–70% in the population), differs from apoE4 (ε4 prevalence 15%–20%) at residue 112, where cysteine in apoE3 is substituted for arginine. ApoE3 differs from apoE2 (ε2 prevalence 8%–10%) at residue 158, where arginine in apoE3 is substituted for cysteine. ApoE4 increases the risk of cardiovascular disease and is associated with increased concentrations of LDL; it also increases risk for Alzheimer disease (AD) and decreases the age of onset and is associated with increased severity or progression of numerous other neurodegenerative disorders. ApoE2 is associated with the genetic disorder type III hyperlipoproteinemia and accelerated atherosclerosis and is “protective” against the development of AD. The structure of apoE and its function in cardiovascular and neurological diseases were elucidated with my distinguished long-term colleagues: Thomas Innerarity, Karl Weisgraber, Stanley Rall, Robert Pitas, Thomas Bersot, and Yadong Huang (Fig. 1) [for reviews, see (1–3)]. My first encounter with the protein that would eventually be known as apoE was at Vanderbilt University Medical School during my MD–PhD studies in 1970. My thesis project was to demonstrate that the liver was the source of plasma VLDL by showing that the particles seen in the rat liver Golgi apparatus were nascent VLDL (the Golgi apparatus is now routinely isolated but then had only been isolated from plant cells by James Morre of Purdue University). The isolated Golgi VLDL resembled plasma VLDL in lipid composition and contained apoB and apoCs—and a minor protein not previously described (4). That minor protein, isolated by Shore and Shore (5) and referred to as the arginine-rich apoprotein, was apoE [for a personal glimpse of these early days, see (6)]. In the late 1960s and early 1970s, the protein constituents of plasma lipoproteins, called apolipoproteins, were being identified and efforts to understand their functions were undertaken (7). My career goal after completing my MD–PhD was to go to the heart institute at the NIH to continue lipoprotein research. Donald S. Frederickson, head of the intramural heart institute and outstanding lipidologist, who knew of my aspiration, called me on Christmas Eve, 1970, and offered me a research position at the NIH. I accepted, but he suggested that it might be helpful if I visited the laboratory first. I did, and upon returning home, my wife Linda asked me about the salary. I had failed to ask! My dream job was completed by assembling an outstanding dedicated research group in 1972–1974. Karl H. Weisgraber, an organic chemist, went on to define the critical structural elements of apoE (e.g., domain interaction). Thomas L. Innerarity, a gifted cell biologist, defined the receptor binding domain of apoE and apoB100. Robert E. Pitas, a lipid biochemist, used his knowledge to ascertain how apoE binds to lipids as model systems. Thomas P. Bersot, a clinical lipidologist, kept us grounded in the clinical relevance of what we were doing. My research at the NIH (1971–1979) focused on alterations in plasma lipoprotein metabolism and atherosclerosis after feeding high-fat, high-cholesterol diets to various animal models, including dogs, rabbits, swine, rats, and marmosets (8, 9). These diets induced the production of atherogenic lipoproteins, called β-VLDL, that were highly enriched in cholesterol and contained large quantities of apoE. Similar apoE-enriched β-VLDL occurred in patients with type III hyperlipoproteinemia. These observations propelled me to want to understand the role of this protein in lipoprotein metabolism. The protein was given its letter designation in 1982 as part of a consensus meeting by investigators who had been studying apoE (10). ApoE was now a bona fide apolipoprotein. LDLs containing only apoB-100 were shown to bind to cell-surface receptors on fibroblasts, known as the LDL receptor (LDLR) (11). The identification of a cholesterol-induced canine lipoprotein with only apoE [apoE-HDL (HDLc)] allowed us to establish the first physiological role for apoE: it is a ligand for LDLR (1–3, 12). ApoE-HDLc floated like HDL but was cholesterol-rich like LDL. The receptor-binding affinity of apoE was more than 20-fold greater than that of apoB100-containing LDL. Arginines and lysines in apoE and apoB-100 were identified as critically important for the interaction of apoE and apoB-100 with the receptors. ApoE was shown